The complement system is part of an innate system that provides an organism a natural defense against microbial agents and infection without the need for a specific antibody. The complement system consists of a number of small plasma proteins, generally synthesized by the liver, and normally circulating as inactive precursors (pro-proteins). When stimulated by one of several triggers (e.g., microbial polysaccharides or lipids, gram-negative bacterial lipopolysaccharides, and surface determinants present on some viruses, parasites, virally-infected mammalian cells, and cancer cells), proteases in the complement system cleave specific proteins to release cytokines and initiate an amplifying cascade of further cleavages. The end-result of this activation cascade is massive amplification of the response and activation of the cell-killing membrane attack complex (MAC). Over 25 proteins and protein fragments make up the complement system, including serum proteins, serosal proteins, and cell membrane receptors. They account for about 5-15% of the globulin fraction of normal human serum.
Three biochemical pathways comprise the complement system: the classical complement pathway, the alternative complement pathway, and the mannose-binding lectin pathway. Once activated, the three pathways generate homologous variants of the protease C3-convertases, which cleave and activate component C3 to generate C3a and C3b fragments and cause a cascade of further cleavage and activation events. As a major effector molecule of the complement system, C3b binds to the surface of foreign substances, cells and/or pathogens, and opsonizes them for destruction. C3b can originate from classical pathway activation and/or from natural spontaneous hydrolysis of C3 via the alternative pathway.
The alternative pathway is triggered by spontaneous C3 hydrolysis directly due to the breakdown of the thioester bond via condensation reaction (C3 is mildly unstable in aqueous environment) to form C3a and C3b. It does not rely on a pathogen-binding antibodies like the other pathways. The resulting C3b binds to the surface of the activating substance. A central reaction that occurs during alternative complement activation is the conversion of the 93 kDa protein called Complement Factor B (CFB) zymogen to an active proteolytic enzyme. This conversion is accomplished in a two-step reaction. In the first step, Factor B forms a magnesium-dependent complex with C3b or C3(H20). The C3(H20)-Factor B complex is formed only in fluid-phase, while the C3b-Factor B complex can be formed either in fluid-phase or on a target surface. In the second step, Factor B is cleaved by Factor D into a 30 to 33 kDa N-terminal fragment “Ba” and a 57 to 60 kDa C-terminal fragment “Bb.” The Ba fragment is released, and the Bb fragment that remains in association with C3b comprises the activation pathway fluid-phase C3 convertase, also known as “C3(H2O)Bb” or “C3bBb.” Properdin (P) stabilizes the C3bBb complex and protects it from decay; thus, another name for the alternative pathway convertase is “C3bBbP.” Although only produced in small amounts, the C3 convertase can cleave multiple native C3 proteins into C3a and C3b. (Christie and Gagnon, 1983, Biochem. J. 209(1):61-70). Cleavage of C3 results in the formation of C3bBb3b, the C5 convertase. This enzyme is also stabilized by P to form C3bBb3bP. C5 convertase can cleave many molecules of C5 into C5a and C5b.
Binding of Ba and Bb fragments to B lymphocyte surface receptors modulates the proliferation of pre stimulated B cells.
The C-terminal half of the Bb fragment contains active site residues characteristic of serine proteases, but has a molecular weight twice that of proteinases previously described, suggesting that it is a novel type of serine proteinase. The Bb portion of the factor B gene is about 4 kb long, and the 3-prime end of the gene codes for amino acids 87-505 of Bb and includes the serine protease domain of the protein (Campbell and Porter, Proc. Natl. Acad. Sci. U.S.A., 1983; 80(14):4464-8).
While the complement system serves as a natural defense system against pathogen substances, the consequences of uncontrolled complement activation can be devastating. Continued activation of the cascade attracts leukocytes which release lysosomal enzymes as a byproduct of phagocytosis, and these lysosomal enzymes can cause necrosis of normal tissue. Alternative complement pathway activation has been implicated as a manifestation of disease or trauma states, and cleavage of Factor B is unregulated in a variety of disease states. In autoimmune diseases, the alternative pathway may contribute directly to tissue damage.
Factor B hyperconsumption and increased catabolism, concomitant with factor B fragment production, occurs in diseases and disorders including gram-negative sepsis, autoimmune diseases, burns, chronic glomerulonephritis, lupus nephritis, systemic lupus erythematosus (SLE), fetal loss in at-risk pregnancy, age-related macular degeneration, rheumatoid arthritis, sickle-cell anemia and several skin diseases. Measurement of alternative pathway activation in vivo has been attempted utilized a number of different techniques to quantitate factor B fragments in biological fluids. Plasma concentrations of factor B fragments, especially Ba fragment levels, in SLE patients showed a positive correlation with disease activity scores. Quantitation of Ba fragment levels in SLE plasma samples are believed to be an accurate reflection of disease activity and Ba is a sensitive predictor of impending flare in these patients. (Kolb, et al., 1989, Complement Inflamm. 6(3):175-204).
Raum et al. (1979) found a rare genetic type of properdin factor B in 22.6% of patients with insulin-dependent diabetes but in only 1.9% of the general population. (Am. J. Hum. Genet., 1979, 31: 35-41).
Highly elevated plasma levels of fragment Ba were found in patients with chronic renal failure (Oppermann, et al., 1991, Kidney Intl., 40:939-947.
As an indication of an acute inflammatory response, an increase in serum levels of C3, C3d and fragment Ba were found in burn patients within one week post-burn. While the assay was very reliable and accurate in monitoring the changes of these indicators in serum, the ELISA assay for C3d and Ba was believed not to be sensitive enough to detect a very low concentration in urine samples from the burn patients, unless a dramatic increase in C3d and Ba were to take place (Wan, et al., 1998, Burns, 24: 241-244).
Animal studies have demonstrated that complement activation is associated with inflammation in the placenta and adverse pregnancy outcomes. Events linked to activation of complement in early pregnancy have been associated with the pathogenesis of pre-eclampsia. In a 2008 study by Lynch, et al., a single plasma sample was obtained from over 701 women before 20 weeks' gestation, and the cohort was followed throughout pregnancy for the development of pre-eclampsia. Elevated levels of the complement activation fragment Bb in plasma at 20 weeks was found to be associated with higher risk of pre-eclampsia. Other significant risk factors for pre-eclampsia included nulliparity, a high body mass index, and maternal medical disease (preexisting maternal hypertension, type 1 diabetes and SLE). Significant risk factors among multiparous women included a history of hypertension in a previous pregnancy and a change of paternity. A model for development of pre-eclampsia was provided, in which complement activation leads to inflammatory events in the trophoblastic tissue and a dysregulation of placental angiogenesis, and when antiangiogenic factors are released (e.g. sFlt1), this causes dysfunction in maternal endothelial cells, leading to pre-eclampsia. Release of Bb into the maternal circulation during the time of uteroplacental vascular remodeling (around 10-20 weeks' gestation) may be a marker of inflammatory events and development of the syndrome later in pregnancy (Lynch, et al., 2008; Am. J. Obstet. Gynecol. 198(4):385.e1-385.e9).
Plasma levels of complement activation fragments and serum levels of angiogenesis-related factors and their interrelationship to obesity and pre-eclampsia were also studied in women between 10 and 15 weeks of gestation. While inflammation in early pregnancy is a significant risk factor for pre-eclampsia, and increased concentrations of Factor Bb were found in pre-eclamptic women, there were limitations of the study due to the small number of women who developed pre-eclampsia, and the conclusion from this study was that complement Bb was not a clinically useful marker for pre-eclampsia (Lynch, et al., B.J.O.G. 2010, 117:456-462).
Pre-eclampsia is a complex multisystem disease that may occur in as many as 10% of pregnancies and poses a potentially significant health risk to the mother and fetus. Pre-eclampsia can lead to spontaneous pre-term birth (SPTB) and mortality. The cause of pre-eclampsia is unclear, and its clinical manifestations can include an aggregate of symptoms, but pre-eclampsia is classically defined as (1) de novo hypertension with proteinuria at 20 weeks of pregnancy or (2) in the absence of proteinuria, gestational hypertension with cerebral symptoms, epigastric or right upper quadrant pain with nausea or vomiting, thrombocytopenia and abnormal liver function tests. However, extensive evidence suggests that this syndrome starts in early pregnancy, and that the immune system plays an important role in the etiology of pre-eclampsia.
While the simultaneous occurrence of high blood pressure and proteinuria after the twentieth week of pregnancy remain the best indicator of pre-eclampsia, some women often do not present these or other symptoms or feelings of illness until the condition becomes severe. Other symptoms include swelling, sudden weight gain, nausea, vomiting, abdominal, shoulder, and lower back pain, headaches, and vision changes. All of these symptoms however are often associated with normal pregnancy, thereby complicating timely diagnosis of pre-eclampsia. Because causes are unknown and indicators before 20 weeks gestation are lacking, once diagnosed, the only treatment for pre-eclampsia is hospitalization for blood pressure monitoring and regular urine sampling, and the only means of completely alleviating pre-eclampsia is Caesarean section or induction of labor (and therefore delivery of the placenta), often pre-term.
Thus, there is a need for convenient, reliable, early detection of or ruling out pre-eclampsia to prevent unnecessary hospitalization of pregnant women who may have high blood pressure and/or proteinuria, but are not likely to develop pre-eclampsia.
Assessment of Factor B cleavage products in urine, plasma or serum can indicate the extent of alternative pathway activation occurring at the time of sample collection. In particular, cleavage product Ba can serve as an indicator of alternative complement pathway activation in pre-eclampsia. A highly specific antibody, having a high negative predictive value, would mean that a negative result (with no fragment Ba detected in a pregnant woman who may or may not be symptomatic or at risk of pre-eclampsia) was very reliable. Such an assay employing a specific antibody to measure the presence or absence of the Ba fragment in urine, less invasive than measurement of complement activation fragment levels in blood, plasma or serum, would provide a valuable means of ruling out pre-eclampsia at an early stage of pregnancy.